Composition comprising pharmaceutical/nutraceutical agent and a bio-enhancer obtained from Glycyrrhiza glabra

ABSTRACT

The invention relates to a new use of a non-alkaloid compound that is plant derived glycoside ‘glycyrrhizin’ as a highly potent bio-enhancer of activity and availability of antibiotics and other drugs including anti-infective and anticancer agents. The molecule of invention facilitates the absorption/uptake of antibiotics and other molecules across the cell membrane in plant and animal cells as well as Gram-positive and Gram-negative bacteria and therefore can be used as a drug facilitator or bioenhancer molecule to increase the affectivity of drugs and nutraceutical agents. The compound having no antimicrobial or cytotoxic activity of its own, is a safe candidate to reduce the drug dosage towards circumventing the problem of drug resistance and the other side effects in anti-infective and anti-cancer therapies.

FIELD OF THE INVENTION

The invention provides novel compositions containing extracts andcompounds obtained from the plant Glycyrrhiza glabra, useful asbio-enhancers and bioavailability facilitators for a variety ofmolecules including anti-infective, anti-cancer agents and nutritionalcompositions. This invention also relates to a method for enhancing thebiological availability, proficiency and effectivity of drugs,pharmaceuticals, nutraceuticals and other related compounds includingamino acids, vitamins and other nutritional elements and ions. Thepresent invention has direct implication in drastically reducing thedosage of antibiotics, drugs and anti-cancer agents while increasing theefficiency of absorption of nutritional elements.

1. Background of the Invention

Since the invention of penicillin in 1928 by Alexander Flemming andtheir wider availability in the 1940s, further variants of existing andnew antibiotics have been on the trial. Compared to the situation in1954 when two million pounds of antibiotics were produced in the US, thepresent figure exceeds 50 million pounds. Antibiotics work either bykilling bacteria (bacteriocidal) or by inhibiting growth(bacteriostatic) and their bioactivities have transformed human abilityto treat many infectious diseases that previously were consideredcertain killers. It is estimated that globally humans consume above 250million doses of antibiotics annually and 20%-50% of that use isunnecessary depending on the class of antibiotic. Further, widespreaduse of antibiotics promotes spread of antibiotic resistance many a timesleading to multiple drug resistance. The total amount of antibiotic inthe given dosage used for treatment of an infection generally is muchhigher than what is actually required to control a given population ofparasite in the infected individual. This is so because all antibioticgiven to the patient in a therapy does not reach the target site. Thismay be due to (i) lower absorption in the gut membrane when taken orally(ii) restrictive uptake by the target microbe or (iii) operation ofefflux pump leading to indiscriminate extrusion of the antibiotics ortherapeutic molecules. Thus, large portions of the drugs we apply arewasted and only a miniscule percentage is being targeted to theinfective microbes. But even worse part is that the unutilizeddrug/antibiotic amount remains as a load in the body and environment.This then acts as a selection pressure, facilitating emergence of drugresistance in parasites leading to their predominance in the niche andultimately resulting into failure of antibiotics against resistantinfections. Additionally, such a situation leads to side effects,illness and reduction in life expectancy being more acute in the olderpopulation. One of the ways that has been feasible to reduce drug dosageis the occurrence of synergism between different therapeutic agents.However, even in such a situation if both the molecules have theantibiotic property, the problem of continued selection pressure onmicrobes is still likely to continue. Therefore, the need is formolecules, which by themselves are not microbicidal but when presentwith a drug or active molecule, enhance its activity and availability(bioenhancers). These molecules by their presence will not exert anyselection pressure for the mutants to emerge resistant against them andon the other hand could reduce the dosage of antibiotics or drugs sothat their ill effects are minimized. This way resistance developmentprocess will be substantially delayed ultimately leading to enhancedlife-span of the novel and existing antibiotics. Such drug/moleculefacilitators should have novel properties like non-toxic to humans,animals or plants, should be effective at a very low concentration in acombination, should be easy to formulate and most importantly enhanceuptake/absorption and activity of the drug molecules. This can lead todevelopment of judicious and strategic concentrations of antibioticswith specific bioenhancers to improve availability of the drug right upto the target for effectively controlling the infectious organisms.

The present invention was the result of planned experiments to provide anovel method for improving activity and bioavailability of antibiotics,drugs and other molecules using a plant glycoside “Glycyrrhizin” indifferent formulations.

The bioavailability of nutrients and enhancement of antibiotics/drugseffectivity is relevant to human, plant as well as animal health andthus the compositions and methods of the invention are also intended tobe used in agriculture and veterinary practice.

2. Description of Related Art

Use of ayurvedic preparation “trikatu” dates back to the period betweenthe seventh century B.C. and the sixth century A.D., which is a Sanskritword meaning three acrids. It refers to a combination of three elementsnamely, black pepper (Piper nigrum Linn.), long pepper (Piper longumLinn.) and ginger (Zingiber officinale Rosc.). It is believed that theuse of “trikatu”, and its constituents individually as well ascollectively, enhances the bioavailability of a number of drugs. Inspecific studies carried out on animals as well as human volunteers, itwas noted that the active component responsible for the increase inbioavailability of various drugs was piperine (U.S. Pat. Nos. 5,616,593and 5,972,382). Till today, thus, the known documented bio-availabilityenhancer is piperine and a series of inventions related to this compoundhave been described by several prior art investigators. Though thecompound piperine has been reported to be enhancing the bioavailabilityof drugs, nutrients and vitamins, still a proper formulation for thecombination is yet to come to the market. Beside this, the compoundpiperine being an alkaloid would find restricted use for the reasons ofits typical action.

The Applicants sought to provide a non-alkaloid molecule capable ofbio-enhancing the availability of anti-infective, nutritional andanti-cancer compounds like ‘taxol’ produced by the yew tree inmicroscopic amounts.

The Applicants screened a large number of the available extracts andknown compounds in the laboratory at the Central Institute of Medicinal& Aromatic Plants, Lucknow, U.P., India (CIMAP), particularly those,which by themselves did not exhibit any antibacterial property. Afterextensive experimentation, it was found that out of 70 pure compoundstested, only one plant compound ‘glycyrrhizin’, a glycoside enhanced thekilling activities of different antibiotics on three diverse kinds ofbacteria including Escherichia coli, Bacillus subtilis and Mycobacteriumsmegmatis. The compound is generally isolated from the roots and stolonof the plant Glycyrrhiza glabra and is a known sweetener andhepatoprotective drug.

Licorice (the extract from the root of the plant) is used to flavorfoods, tobacco, drinks, medicines and sweets. It has expectorant actionto treat bronchitis and can reduce inflammation, allergies, asthma,gastritis, peptic ulcers, rheumatism, and sore throat. It helps theliver to detoxify drugs and is used for treatment of liver disease.Licorice strengthens the immune system, stimulates the adrenal gland,and is diuretic and laxative. Licorice from roots of Glycyrrhiza glabra,contains glycyrrhizin, which is 50 times sweeter than sugar. Primaryuses for licorice include treatment for peptic ulcers and stomachailments, respiratory and intestinal passages. Licorice makes awonderful expectorant expelling mucus from the chest area.

The compound when studied by the Applicants also showed enhancement inthe activity of known azole class antifungal agents like clotrimazoleagainst infectious fungal strain of Candida albicans. More important wasthe obvious enhancement in the cell division inhibitory activity againstthe breast cancer cell line MCF-7.

Accordingly, the Applicants have sought to increase the absorption ofanti-infective, nutritional and anti-cancer compounds by the body andincrease their utilization by the body by providing novel compositionscontaining extracts from the plant glycyrrhiza glabra.

OBJECTS OF THE INVENTION

The main object of the invention is to provide novel compositionscomprising extracts and compounds from the plant Glycyrrhiza glabrauseful as a bioenhancer and capable of improving or enhancing thebio-availability of drugs such as antibiotics, anti-infective agents andof nutritional compounds.

Another object of the invention is to provide compositions comprisingextracts and compounds from the plant Glycyrrhiza glabra useful asbio-enhancers and bioavailability facilitators, thus enabling effectivetransport of drugs and molecules across biological membranes to reachtarget sites.

Still another object is to provide methods for enhancing thebio-availability of drugs molecules and nutritional to the target sitein the body.

SUMMARY OF THE INVENTION

Accordingly, the invention provides novel compositions containingextracts and compounds from the plant Glycyrrhiza glabra useful asbio-enhancers and bio-availability facilitators for drugs and moleculessuch as antibiotics, anti-infective agents, anti-cancer agents and fornutritional compounds. The invention also provides methods for enhancingthe availability of drugs, molecules and nutritional compounds toeffectively reach the target site in the body.

DETAILED DESCRIPTION

Accordingly, the invention provides a composition comprising aneffective amount of an extract or compound obtained from the plantGlycyrrhiza glabra and useful as a bio-enhancer and bioavailabilityfacilitator together with a therapeutically effective amount of one ormore nutraceuticals, antibiotics, anti-infective agents and anti-canceragents.

In an embodiment, the composition contains an extract from the leaves,stem, roots or stolon of Glycyrrhiza glabra, glycyrrhizic acid orglycyrrhizin, or a combination thereof

In still another embodiment, glycyrrhizin and glycyrrhizic acid areobtained from the plant Glycyrrhiza glabra.

In yet another embodiment, the anti-infective agents are selected fromanti-bacterial, anti-fungal and anti-tuberculosis agents.

In yet another embodiment, the extract from the roots of Glycyrrhizaglabra is licorice. In an embodiment, the compound as bioavailabilityenhancer facilitates the transport of the compound(s) across thebiological membranes to reach the target site for bettereffectivity/activity.

In an embodiment, the concentration of glycyrrhizin ranges from 0.05 to50% of the weight of the anti-bacterial compounds.

In still another embodiment, the concentration of glycyrrhizin rangesfrom 0.10 to 10% of the weight of the nutraceutical compounds.

In yet another embodiment, the concentration of glycyrrhizin ranges from0.25 to 20% of the weight of the anti-fungal agents.

In another embodiment, the anti-bacterial agent is selected from thegroup consisting of β-lactams, macrolides, quinolones,fluoro-quinolones, aminoglycosides, glycopeptides, rifamycins, folateinhibitors, tetracyclines, anti-tuberculosis agents and other similarmicrobicidal compounds.

In still another embodiment, the anti-fungal agent used is selected fromthe group comprising of polyene, triazole, imidazole, clotrimazole andother fungicidal compounds. In still another embodiment of the presentinvention bioenhancer improves the uptake of anti-infectives, anticanceragents and other molecules when glycyrrhizin is provided prior to thetreatment with these molecules.

In yet another embodiment of the present invention the anticancer agentsinclude paclitxel (taxol) or other cell division inhibitors controllingthe growth and multiplication of tumor/cancer cells.

In yet another embodiment of the present invention living system used isselected from bacteria, fungi or any living cells.

In yet another embodiment of the present invention the bioenhancerelement is used in pure form as glycyrrhizin or as precursor/derivativeslike glycyrrhizic acid.

In yet another embodiment of the present invention the bioenhancerelement is administered provided as licorice in form of aquous/alcoholicextracts, decoction or direct root flakes/chips/sticks through wettingby any means.

In yet another embodiment of the present invention a pharmaceuticalcomposition to increase the bioactivity and bioavailability comprisingof the plant compound glycyrrhizin and a bio-active chemical compoundselected from the group consisting of anti-bacterial, anti-fungal,anti-cancerous and other similar compounds including nutraceuticalswherein, the glycyrrhizin concentration ranges from 0.10 to 10% of theweight of the nutraceuticals compound used.

In yet another embodiment, the anti-bacterial agent is selected from thegroup consisting of β-lactams, macrolides, quinolones,fluoro-quinolones, aminoglycosides, glycopeptides, rifamycins, folateinhibitors, tetracyclines, anti-tuberculosis agents and other similarmicrobicidal compounds wherein, the glycyrrhizin concentration rangesfrom 0.05 to 50% of the weight of the anti-bacterial compound used.

In yet another embodiment, the macrolides comprise erythromycin,clarithromycin, azithromycin and other similar compounds.

In yet another embodiment, the quinolones and fluoroquinolones used areselected from the group consisting of nalidixic acid, norfloxacin,ciprofloxacin, sparfloxacin and similar other compounds.

In yet another embodiment of the present invention the aminoglycosidesused are selected from the group consisting of streptomycin, gentamicin,amikacin and other similar compounds.

In yet another embodiment of the present invention the glycopeptidesused are vancomycin, teicoplanin and other similar compounds.

In yet another embodiment of the present invention the rifamycins usedare rifampicin and other similar compounds.

In yet another embodiment of the present invention the anti-tuberculosisagents used are isoniazid, pyrazinamide, ethambutol and other similarcompounds.

In yet another embodiment of the present invention the folate inhibitorsare trimethoprim; sulphonamides and other similar compounds.

In yet another embodiment, the β-Lactams comprise penicillin,flucloxacillin, cloxacukkub, methicillin, cephalosporins, carbapenenmsand similar compounds.

In yet another embodiment, other similar microbicidal compounds arestreptogramins and oxazolidinones.

In another embodiment the anti-fungal agent is selected from the groupconsisting of polyene, triazole, imidazole, clotrimazole and otherfungicidal compounds wherein, the glycyrrhizin concentration ranges from0.25 to 20% of the weight of the anti-fungal compound used.

In yet another embodiment the polyenes used are amphoterecin B, nystatinand other similar compounds.

In yet another embodiment the triazoles used are fluconazole,itraconazole and other similar compounds.

In yet another embodiment the imidazoles used are ketoconazole and othersimilar compounds.

In yet another embodiment other fungicidal compounds are griseofulvinand terbinafine.

In yet another embodiment, the anti-cancer agent is selected from thegroup consisting of Paclitaxel (Taxol), Docetaxel (Taxotere),Vinblastine (Velban), Vincristine (Oncovin), Vinorelbine (Navelbine) andother similar compounds used in cancer therapy.

In another embodiment, the levels of glycyrrhizin ranges from 10 to10,000 folds of the weight of the anti-cancer compound used.

In another embodiment, the nutraceuticals used to increase thebio-activity and bio-availability are selected from the group consistingof vitamins, amino acids, hormones and other nutritional supplements.

In yet another embodiment, the pharmaceutical compositions comprising ofthe plant compound glycyrrhizin in form of liquorice/dried root with thebioactive chemical compound(s) to increase their bioactivity andbioavailability wherein, the glycyrrhizin in form of liquoricepreparation or dried root is given as one gram equivalent to 40 mg ofglycyrrhizin.

The invention also provides a method for enhancing the bio-availabilityof herbal extracts, anti-infective agents, anti-cancer and nutraceuticalcompounds/compositions across biological membranes in living systems;said method comprising the steps of administering to a subject in needthereof a composition comprising an effective amount of the bioenhancerobtained from the plant Glycyrrhiza glabra, and any one ofanti-infective agents, anti-cancer agents and nutraceutical compounds.

In an embodiment, the bio-enhancer is selected from the extract ofGlycyrrhiza glabra, glycyrrhizin, glycyrrhizic acid obtained fromGlycyrrhiza glabra or combinations thereof

In yet another embodiment, administration of the composition circumventsthe side effects of chemotherapy by substantially reducing the dosagesof therapeutic agents comprising of anti-bacterial, anti-fungal andanti-cancerous agents wherein the side effects of those agents can bebone marrow suppression, liver and kidney toxicity, pulmonary scarring,high fevers, skin reactions, Nausea and vomiting, hair loss, skin rash,mouth sores, diarrhoea, loss of appetite and confusion, lethargy andambulation problems (ataxia), anaphylactic reactions (consisting of fastheart rate, wheezing, lowered blood pressure and facial edema,cerebellar dysfunction (consists of slurring of speech, walkingproblems, as well as eye motion problems), loss of periods (menses),ovarian failure can occur, painful soft tissue ulcers, cardiotoxicity,hypersensitivity reactions, abnormal blood pressure, sweating, bleeding,shortness of breath and many related problems.

In still another embodiment, the bioenhancer improves the uptake ofanti-infectives, anticancer agents and other molecules when glycyrrhizinis provided prior to the treatment with these molecules.

The invention also provides use of the extract or the compound obtainedfrom the plant Glycyrrhiza glabra, as a bioenhancer and bioavailabilityand facilitator of nutritional compounds and drugs and moleculesselected from anti-infective and anti-cancer agents.

The invention is described in detail herein below with reference to thefollowing drawings wherein:

FIG. 1: represents the structural formula of Glycyrrhizic acid; and

FIG. 2: represents the structural formula of Glycyrrhizin

Some of the embodiments of the invention are illustrated herein below bythe following bioassays and examples which are provided merely toillustrate the invention and should not be construed as limitations onthe inventive concept embodied herein.

The methodology followed for screening of this compound (Glycyrrhizicacid and Glycyrrhizin) included specifically designed bioassays asfollows:

1. Assay for Bio-Enhancement of Anti-Infective Agents

-   a) The minimum inhibitory concentration (MIC) of any antibiotic or    drug molecule is determined against Escherichia coli (ATCC 10536),    Bacillus subtilis (ATCC 6015 ) and Mycobacterium smegmatis    (ATCC 14468) and Candida albicans (ATCC 10231) in broth and disc    diffusion assays.-   b) The antibiotics or antifungal agents at concentrations 1/10, ⅛,    ⅙, ¼, ⅓, ½ and equal to MIC are added alone and in combination with    the test compound at varying concentrations in broth assays to    evaluate the comparative level of inhibition and similarly at    varying concentrations in disc diffusion assay.-   c) The combinations showing enhancement in terms of higher activity    than antibiotic alone for the inhibition of bacterial growth (large    inhibition zone in disc diffusion and higher killing at lower    concentration in broth assay) are picked up for future testing.

d) In broth assays the activity is quantified by counting number ofviable cells in a given treatment and converted in fold enhancement bythe combination treatment compared to antibiotic/drug alone in thekilling percentage of cells.

e) The pretreatment assay is followed to determine whether thebioenhancer which may be either an extract obtained from the plantGlycyrrhiza glabra, glycyrrhizin or glycyrrhizic acid, is required alongwith antibiotic to enhance its activity or even its withdrawal aftertreatment or prior to antibiotic treatment would benefit. For this, thecells are treated with the bioenhancer/compound for 4 to 8 hours andthen washed free of it by centrifugation and washing in sterile water.This is followed by treatment with antibiotic/drug as in steps (b) to(d) above.

f) The results of the bio-assays carried out are represented in thetables provided hereinbelow wherein tables 1-4 represent the activity ofglycyrrhizin. Tables 7-9 depict the activity of glycyrrhizic acid andtables 10-11 depict the activity of licorice.

2. Assay for Bio-Enhancement of Anti-Cancer Agents

-   a) MCF-7 (Breast cancer cell line is inoculated at a density of    about 0.1×10⁶ cells in MEM medium in the wells of 24 well plate.-   b) The used up medium in these cultures is replaced with fresh    medium after 18 hours in each well.-   c) The test compound (s) is added at desired concentrations in    different wells just after the medium replacement.

d) Observations are recorded on the cell count after 36 hours for whichthe following steps are required.

-   -   i. The medium is removed from the wells.    -   ii. The wells are rinsed with 1 ml PBS (Phosphate buffer        saline).    -   iii. To each well 500 μl of freshly prepared trypsin solution        (0.1% in PBS) is added.    -   iv. Trypsin solution is removed after 30 seconds and the plate        is gently tapped till the cells are released from the plate        surface.    -   v. Fresh 1 ml of MEM growth medium is added and agitated with a        pipette to obtain a cell suspension.    -   vi. 10 μl of cell suspension is taken on the haemocytometer and        a cover glass is placed over the counting chamber.    -   vii. The number of viable cells is counted in 5 big squares and        the reading is taken from 5 microscopic fields to determine the        average.

viii. The cell count (titre per ml) in the original sample is thencalculated as average count×10³. Composition of Minimum Essential Medium(MEM): 100 ml MEM powder (Sigma - Aldrich, USA) = 0.96 g HEPES Buffer(Sigma - Aldrich, USA) = 0.26 g Sodium Bicarbonate = 0.22 g Penicillin G= 10 mg Streptomycin = 20 mg Gentamycin = 5 mg Foetal Calf Serum = 15 mlFoetal Calf Serum = 15 ml Distilled water = 85 ml3. Bioavailability Tests Through Biological Membrane

-   a) A specially designed U-tubes of glass consisting of two component    (opposite-L type) were used in which one open end of a L-shaped was    tapered to fit within the untapered end of the other L-tube.-   b) The membrane of goat gut (initial part) was stretched and fixed    to act as the barrier between the two ends such that by joining the    two L-tubes, a U-tube was made.-   c) Sterile distilled water was then filled in both the sides to    equal height/level.-   d) The antibiotic/compound was added to the donor tube (tapered) and    through spectro-photometer the transfer of molecule was observed    using UV and visible absorption maxima of the respective molecules    by taking the OD at defined wavelengths.

EXAMPLES

In the next step of elucidation of the enhancer action the applicantsexperimented with the killing activities of different antibioticsagainst the bacteria singly and in combination with the test compoundglycyrrhizin following the method described above. These experiments areto be considered with assay for bioenhancement of the compoundsglycyrrhizin/glycyrrhizic acid and licorice, as described earlier. Whenthe bacteria were grown in presence of the compound as such nosignificant killing was observed. In all the experiments theglycyrrhizin concentration was kept at 1 μg/ml, unless it isspecifically mentioned.

Example 1 Glycyrrhizin Mediated Enhancement in the Killing Action ofAntibiotics Against Gram-Negative Bacterium Escherichia coli

TABLE 1 Survival Survival fraction *Folds fraction of viable cellsenhancement of viable cells upon treatment in Concen- upon treatmentwith antibiotic- antibiotic tration with antibiotic glycyrrhizinactivity Antibiotics μg/ml alone (a) combination (b) (a/b) Rifampicin 100.42 0.11 3.8 Rifampicin 20 0.28 0.02 14.0 Nalidixic 8 0.19 0.02 9.5acid Tetracycline 2 0.42 0.22 1.9 Tetracycline 4 0.27 0.03 9.0Ampicillin 6 0.38 0.03 12.6 Ampicillin 8 0.013 0.006 2.1

Example 2 Glycyrrhizin mediated enhancement in the killing action ofantibiotics against Gram-positive bacterium Bacillus subtilis

TABLE 2 Survival Survival fraction fraction of of viable cells *Foldsviable cells upon treatment enhancement Concen- upon treatment withantibiotic- in antibiotic tration with antibiotic glycyrrhizin activityAntibiotics μg/ml alone (a) combination (b) (a/b) Nalidixic 6 0.50 0.114.50 acid Nalidixic 8 0.27 0.04 6.75 acid Tetracycline 1 0.10 0.02 5.00

Example 3 Glycyrrhizin Mediated Enhancement in the Killing Action ofAntibiotics Against Bacterium Mycobacterium smegmatis

TABLE 3 Survival Survival fraction fraction of of viable cells *Foldsviable cells upon treatment enhancement Concen- upon treatment withantibiotic- in antibiotic tration with antibiotic glycyrrhizin activityAntibiotics μg/ml alone (a) combination (b) (a/b) Rifampicin 0.5 0.56 ×10⁻⁴ 0.086 × 10⁻⁴ 6.5 Nalidixic 4 0.70 0.12 7.7 acid Tetracycline 0.50.3 0.2 1.5

From the above experiments it was deduced that the potency of theantibiotic is increased when applied along with the compoundglycyrrhizin. It was interesting to note that the same results wereobserved when the bacterial cells were pretreated with glycyrrhizin for4-8 hours and then the cells were washed free of the glycyrrhizin bycentrifugation and resuspending the cells in fresh medium followed bytreatment with respective antibiotics. This implicated some changesinduced by glycyrrhizin on the bacterial surface/membrane making it morereceptive to the antibiotics and hence enhancing bioavailability andultimately activity/bactericidal effect of the given antibiotic.

Example 4 Glycyrrhizin Mediated Enhancement Observed in Disc DiffusionAssays for the Killing Action of Antifungal Agent Against Candidaalbicans

TABLE 4 Clotrimazole Glycyrrhizin Zone of inhibition (μg/disc) (μg/disc)(mm) 0 0 0 0 1 0 4 0 7 4 1 11

Example 5 Glycyrrhizin Mediated Enhancement in the Activity ofAnticancerous Agent “Taxol”

TABLE 5 Final titre of Final titre of viable cells Taxol Initial titreviable cells upon treatment with Concentration of viable upon treatmenttaxol-glycyrrhizin (μg/ml) cells with taxol alone combination 0.01 0.1 ×10⁻⁶ 0.05 × 10⁶ 0.025 × 10⁶ 0.02 0.1 × 10⁻⁶ 0.04 × 10⁶ 0.024 × 10⁶ 0.050.1 × 10⁻⁶ 0.03 × 10⁶ 0.021 × 10⁶ Without taxol Final titre Control 0.1× 10⁻⁶ 0.16 × 10⁻⁶ Control 0.1 × 10⁻⁶ 0.12 × 10⁻⁶ (glycyrrhizin)

Example 6 Glycyrrhizin Mediated Enhancement in the bioavailabilityAcross the Biological Membrane

TABLE 6 OD measured as Absorbance Wave (specific to the compound maxima)Compound(s) in length across the membrane in receiving tube after thedonor tube (nm) 1 hr 2 hr 3 hr 4 hr 5 hr 6 hr Rifampicin A₃₄₀ 0.00390.0275 0.0371 0.0440 0.0906 0.1165 A₄₇₅ 0.0015 0.0148 0.0210 0.02970.0334 0.0394 Rifampicin + Glycyrrhizin A₃₄₀ 0.0070 0.0638 0.1465 0.20900.3424 0.3930 A₄₇₅ 0.0100 0.0395 0.0575 0.1362 0.2365 0.2853 VitaminB-12 A₃₆₀ 0.0250 0.0556 0.0714 0.1283 0.1370 0.1595 A₅₃₀ Vitamin B-12 +Glycyrrhizin A₃₆₀ 0.0582 0.0627 0.1058 0.2051 0.2616 0.2984 A₅₃₀

Here, the applicants observed glycyrrhizin-mediated enhancement in thecell division inhibitory action of anti-cancer agent taxol in the animalcell culture experiments using cancerous cell line MCF-7. As evident inTable 5 above, the anticancerous activity of ‘Taxol’ in terms ofinhibiting the growth and multiplication of MCF-7 cancer cells wasmarkedly enhanced by at least 5 folds. In these experiments, thecancerous cells growth inhibition by Taxol (@ 0.01 μg/ml) in presence ofglycyrrhizin (@ 1 μg/ml) was higher than even the treatment with taxol(@ 0.05 μg/ml) alone.

Example 7 Glycyrrhizic Acid Mediated Enhancement in the Killing Actionof Antibiotics Against Escherichia coli in Broth Assay

TABLE 7 Survival fraction Survival of viable cells *Folds fraction upontreatment en- of viable cells with antibiotic hancement Concen- upontreatment glycyrrhizic acid in tration with antibiotic combinationantibiotic Antibiotics μg/ml alone (a) (b) activity (a/b) Rifampicin 200.57 0.281 2.03

TABLE 8 Glycyrrhizic acid mediated enhancement in the killing action ofantibiotics against Escherichia coli in disc diffusion assay.Glycyrrhizic Net zone of Rifampicin (μg/disc) acid (μg/disc) inhibition(mm) 0 0 0 0 4 0 500 0 2 500 4 3

TABLE 9 Glycyrrhizic acid mediated enhancement in the killing action ofantibiotics against Mycobacterium smegmatis in disc diffusion assay.Rifampicin Glycyrrhizic Net zone of (μg/disc) acid (μg/disc) inhibition(mm) 0 0 0 0 4 0 0.4 0 3 0.4 4 4

In the above experiments, the Applicants used Glycyrrhizic acid to testthe potency of this acid in enhancing the activity of antibiotics. TheApplicants observed that the killing action the antibiotics (such asRifampicin) is enhanced when used with Glycyrrhizic acid. Table 7 showsthat when the cells were treated with Rifampicin alone, the survivalfraction was about 0.57. However, the survival fraction of cells whentreated with a combination of rifampicin and glycyrrhizic acid was muchless i.e. about 0.281. This shows that the combination of glycyrrhizicacid with rifampicin enhances the availability of rifampicin to thecells. Similarly, the results of the disc diffusion assay also show thatthe combination of glycyrrhizic acid with rifampicin is more effectiveagainst mycobacterium smegmatis than glycyrrhizic acid or rifampicinused singly.

Example 8 Licorice (25 (μg/ml) Mediated Enhancement in the KillingAction of Rifampicin Against Gram-Negative Bacterium Escherichia coli

TABLE 10 Survival Survival fraction *Folds fraction of of viable cellsen- viable cells upon treatment hancement Concen upon treatment withantibiotic in antibiotic tration with antibiotic glycyrrhizic acidactivity Antibiotics μg/ml alone (a) combination (b) (a/b) Rifampicin 100.420 0.135 3.10

TABLE 11 Licorice (25 μg/ml) mediated enhancement in the killing actionof antibiotics against Gram-positive bacterium Bacillus subtilus.Survival Survival fraction fraction of of viable cells viable cells upontreatment *Folds upon treatment with antibiotic enhancement inConcentration with antibiotic glycyrrhizic acid antibiotic activityAntibiotics μg/ml alone (a) combination (b) (a/b) Nalidixic acid 6 0.5000.121 4.10

Tables 10 and 11 show the bio-enhancing effects observed in the use oflicorice, the extract from the roots of Glycyrrhiza glabra. As can beseen from Tables 10, the survival rate of bacteria treated with theantibiotic Rifampicin is more than when treated with a combination ofRifampicin and licorice. Similarly, Table 11 shows that the survivalrate of the bacteria treated with nalidixic acid is less than whentreated with a combination of nalidixic acid and licorice. The resultsin Tables 10 and 11 demonstrate, the use of licorice as an effectivebio-enhancer and bioavailability facilitator.

In the experiments related to transport across the mammalian gutmembrane, it was observed that the compound glycyrrhizin enhances thetransport of antibiotics e.g. Rifampicin, Tetracycline, Nalidixic acid,Ampicillin and vitamins like B-1 and B-12 across the gut as well asartificial membrane. The enhancement in transport is approximately 2 to6 folds.

Several medicinal plants are cited for use in Indian system of medicinefor various activities such as immunostimulant, adaptogenic, anticancer,antimalarial, antimicrobial, angiogenetic, hepatoprotective,hypolinidemic, hypoglycemic, memory enhancing etc. The genus Glycyrrhiza(Mulethi) has attracted sizable importance for compound(s) withhepatoprotective ability. In Glycyrrhiza glabra (family, Leguminosae) 12different varieties (Biovars) are known. Among these only three namelytypica, glandilifera and violacea are commonly used for thepharmaceutical and confectionary requirements. Glycyrrhiza glabra var.typica is the source of Spanish Liquorice and is sweeter than othertypes. Glycyrrhiza glabra var. glandilifera is the source of Russianliquorice, which is moderately sweet and some what bitter, also more orless acrid. Glycyrrhiza glabra var. violacea is known as the Persian orIranian liquorice of commerce and has thick roots. The name Glycyrrhiza(popularly known as mulethi in India) is of Greek origin meaning sweetroot. Cultivation of this species is mostly limited to US, EasternEurope and several CIS of former USSR and scattered in western andnorthern India. The roots of the plant Glycyrrhiza glabra are highlyuseful as these contain the important glycoside Glycyrrhizin for itshepatoprotective ability. The root extracts also contain flavonoids,saponins, sterols, amino acids, gums essential oils, sugars andstarches. The plant takes at least 4-5 years to achieve substantialproduction of glycyrrhizin in the roots. Generating sufficient plantingmaterial for commercial cultivation is thus desirable for harvestingrequired quantity of Glycyrrhizin on sustainable basis.

This molecule (Glycyrrhizin) is a known sweetener and hepatoprotectivedrug. It helps the liver to detoxify drugs and is given for liverdisease. The molecule is known to be strengthening the immune system,stimulates the adrenal gland, and is diuretic and laxative. This is alsoused to treat peptic ulcers and stomach ailments, respiratory andintestinal passages. Considering the wide use of the molecule intraditional system of medicine and the level of toxicity well tested,this can be used along with the antibiotics and other molecule ofimportance to enhance the efficacy on the target. This molecule(glycyrrhizin) in the Applicant's study facilitates the transport/uptakeof various compounds including antibiotics, antifungal and anticanceragents, vitamins and amino acids etc. across the membrane therebyincreasing the absorption or bioavailability various biological systemsincluding bacteria, animal and plant cells. This thus has immenseimportance for absorption of the drugs, pharmaceuticals, nutraceuticaland other related compounds and ions by the cells and hence hugepotential of being used as drug/molecule facilitators.

Great emphasis now is being laid towards quality assurance of crudedrugs from plants sources widely used in the Indian system of medicine.The scientific study of traditional medicines, derivation of drugsthrough bioprospection and systematic conservation, domestication andcultivation of the concerned medicinal plants has assumed greatimportance in the present day context when more and more people prefersafe and effective medicines at affordable price for curing theirailments. The present invention enlarges the scope and use of thenatural glycoside glycyrrhizin in therapeutical and nutraceuticalapplication. These plants synthesizing multi-use molecules areGlycyrrhiza sps and Abarus sps. The preferred plant is Glycyrrhizaglabra which is propagated through underground rhizome. This demandslarge amount of planting material which may be possible by micropropagation in vitro for its commercial cultivation. Agrotechnology andsuitable cultivars for its cultivation in India are therefore requiredthrough screening of germplasm and genetic selection. At CIMAP agenetically superior variety of Glycyrrhiza glabra var. typica named as‘Misree’ is available for cultivation with better yield of glycyrrhizin(Kukreja. A K, Tyagi B R, Muni Ram and Sushil Kumar (1997) Liquoricecultivar—Mishree. Journal of Medicinal and Aromatic Plants Sciences. 19:732-733).

Procedure for isolation of quality glycyrrhizin the main compound fromthe root extract. Of the plant Glycyrrhiza glabra var. typica was alsostandardized. For this purpose we choose the plant Glycyrrhiza glabravar. typica ‘Misree’ (the released variety of CIMAP) and commerciallyavailable plant material. Fresh roots were harvested from the plant andshade dried. Commercial dried roots were purchased from the market. Theprotocol followed for the isolation was as follows:

Protocol:

-   -   1. Plant material (250 gram) was dipped in 900 ml of Butanol:        glacial acetic acid ethyl alcohol (1: 1:1) for 48 hours.    -   2. This was filtered through filter paper and concentrated in        vacuums rota-vapour.    -   3. The solid material thus obtained was dissolved in acetic acid        (180 ml) and treated with diethyl ether 2 litre (10 times, each        with 200 ml) to remove impurity as precipitate which was        filtered out.    -   4. Then the liquor in diethyl ether was evaporated to 250 ml and        fractionated through liquid—liquid fractionation twice in water,        followed by discarding the water fraction.    -   5. This diethyl ether fraction was further concentrated in        vacuum rotavapour to obtain the compound.    -   6. The compound was hydrolyzed with 80 ml of NaOH (NaOH 5% in        Methaol) at 80°-90° C for 1 hour.    -   7. The hydrolyzed reaction mixture was acidified (100 ml of        glacial acetic acid acetic acid and 5 ml of concentrated HCl) to        pH 3.6 to form a ppt which was filtered and washed with ethanol

After isolation, the compound was checked for the purity compared to thestandard glycyrrhizin obtained from Sigma chemical Inc.

Preparation of the test solution: One gram of the isolated compound wasextracted in 20 ml chloroform for 15 min. After filtration the filtratewas discarded. The marc was refluxed for 1 hour with 30 ml of 0.5 MH₂SO₄. The unfiltered mixture was then cooled and extracted withchloroform (2×20ml) and the combined CHCl₃ extract was concentrated. Theresidue was dissolved in 1.0 ml CHCl₃: Methanol (1:1) mixture.

Reference solution: Five milligram of glycyrrhizin was refluxed with 20ml 0.5 M H₂SO₄. This was cooled and extracted with chloroform (2×10 ml).The combined CHCl₃ extract was evaporated and the residue was dissolvedin 1.0 ml CHCl₃: Methanol (1:1) mixture.

Solvent system: Toluene: ethyl acetate: glacial acetic acid (12.5:7.5:0.5)

Procedure: The test and reference solutions were applied in twodifferent tracks on a precoated silica gel GF₂₅₄ plate (5×15 cm) ofuniform thickness (0.2 mm). The plate was developed in the solventsystem to a distance of 12 cm.

Scanning: The plate was scanned densitometrically at 254 nm in both thetracks and the fingerprint profiles were recorded.

Visualization of Spots:

-   (i) Under UV 254 nm.-   (ii) The plate was sprayed with anisaldehyde-sulfuric acid reagent    and heated at 110° C. for 5-10 min.    Evaluation:-   (i) Under UV 254 nm light (before spraying):one spot (0.41) was    visible in the sample solution track, corresponding to (Rf 0.41)    glycyrrhetic acid of reference track.-   (ii) In daylight (after spraying): Gylcyrrhetic acid was visible as    a dark violet spot in both reference and test solution tracks.

The above experiments indicate a sound procedure of isolating fairlypure glycyrrhizin from the root of the plant variety ‘Misree’ of CIMAPas well as market available dry root samples. By this procedure about0.1 to 0.25% of glycyrrhizin could be isolated. The protocol can bescaled up or scaled down depending upon the initial starting material.

Advantages

-   1. The plant extract of Glycyrrhiza glabra and glycyrrhizin in the    Applicant's study facilitates the transport/uptake of various    compounds including antibiotics, antifungal and anticancer agents,    vitamins and amino acids etc. across the membrane thereby increasing    the absorption or bioavailability various biological systems    including bacteria, animal and plant cells. This thus has immense    importance for absorption of the drugs, pharmaceuticals,    nutraceutical and other related compounds and ions by the cells and    hence huge potential of being used as drug/molecule facilitators.-   2. The present invention enlarges the scope and use of the natural    glycoside glycyrrhizin in therapeutical and nutraceutical    application.-   3. Glycyrrhizin is nontoxic to humans,animals or plants, is    effective at very low concentrations in a combination,easy to    formulate and enhances the uptake/absorption and activity of drug    molecules. Thus the novel use of this compound as described in the    present invention is safe and effective for bioenhancement of    activity of drugs.-   4. Glycyrrhizin by its presence will not exert any selection    pressure for the mutants to emerge resistant against them and reduce    the dosage of antibiotics or drugs so that their ill effects are    reduced.

1. A composition comprising an effective amount of an extract orcompound obtained from the plant Glycyrrhiza glabra and useful as abio-enhancer and bioavailability facilitator together with atherapeutically effective amount of one or more nutraceuticals,antibiotics, anti-infective agents and anti-cancer agents. 2-4.(canceled)
 5. A composition as claimed in claim 1 wherein theanti-infective agents are selected from anti-bacterial, anti-fungal andanti-tuberculosis agents.
 6. (canceled)
 7. A composition as claimed inclaim 1 wherein the concentration of glycyrrhizin ranges from 0.10 to10% of the weight of the nutraceutical compounds.
 8. A composition asclaimed in claim 1 wherein the concentration of glycyrrhizin ranges from0.25 to 20% of the weight of the anti-infective agent.
 9. A compositionas claimed in claim 5 wherein the anti-bacterial agents are selectedfrom the group comprising P-lactams, macrolides, quinolones,fluoro-quinolones, aminoglycosides, glycopeptides, rifamycins, folateinhibitors, tetracyclines, anti-tuberculosis agents and microbicidalcompounds.
 10. A composition as claimed in claim 9 wherein thequinolones and fluoro-quinolones are selected from the group consistingof nalidixic acid, norfloxacin, ciprofloxacin, sparfloxacin and similarother compounds.
 11. A composition as claimed in claim 9 wherein theβ-Lactams are selected from the group comprising penicillin,flucloxacillin, cloxacukkub, methicillin, cephalosporins andcarbapenenms.
 12. A composition as claimed in claim 9 wherein themacrolides are selected from erythromycin, clarithromycin, azithromycinand other similar compounds.
 13. A composition as claimed in claim 9wherein the aminoglycosides are selected from the group consisting ofstreptomycin, gentamicin, amikacin and similar compounds.
 14. Acomposition as claimed in claim 9 wherein the glycopeptides are selectedfrom vancomycin, teicoplanin and other similar compounds.
 15. Acomposition as claimed in claim 9 wherein the rifamycins are rifunpicinand similar compounds.
 16. A composition as claimed in claim 9 whereinthe folate inhibitors are selected from trimethoprim, sulphonamides andother similar compounds.
 17. A composition as claimed in claim 9 whereinthe microbiocidal compounds are selected from streptogramins andoxazolidinones.
 18. A composition as claimed in claim 5 wherein theanti-tuberculosis agents are selected from the group comprisingisoniazid, pyrazinamide, ethambutol and other similar compounds.
 19. Acomposition as claimed in claim 5 wherein the anti-fungal agents areselected from the group consisting of polyene, triazole, imidazole,clotrimazole and other fungicidal compounds.
 20. A composition asclaimed in claim 19 wherein the polyene is selected from amphoterecin B,nystatin and other similar compounds.
 21. A composition as claimed inclaim 19 wherein the triazole is selected from fluconazole, itraconazoleand other similar compounds.
 22. A composition as claimed in claim 19wherein the imidazole is selected from ketoconazole and other similarcompounds.
 23. A composition as claimed in claim 19 wherein thefungicidal compound is selected from griseofulvin and terbinafine.
 24. Acomposition as claimed in claim 1 wherein the anti-cancer agent isselected from the group consisting of paclitaxel, docetaxel,vinblastine, vincristine, and vinorelbine.
 25. A composition as claimedin claim 1 wherein the concentration of giycyrrhizin ranges from 10 to10,000 fold folds of the weight of the anti-cancer compound.
 26. Acomposition as claimed in claim 1 wherein the nutraceutical compoundsare selected from the group consisting of vitamins, amino acids,hormones and other nutritional supplements.
 27. (canceled)
 28. A methodfor enhancing the bio-availability of a herbal extract, ant-infectiveagent, anti-cancer agent or nutraceutical compound across biologicalmembranes in a patient in need thereof comprising administering to thepatient composition comprising an effective amount of a bioenhancerobtained from the plant Glycyrrhiza glabra, and any one of ananti-infective agent, anti-cancer agent or nutraceutical compound.
 29. Amethod as claimed in claim 28 wherein the bio-enhancer is selected fromthe extract of Glycyrrhiza glabra, glycyrrhizin, glycyrrhizic acidobtained from Glycyrrhiza glabra or combinations thereof.
 30. A methodas claimed in claim 28 wherein the bioenhancer is obtained from theroots, stem, stolon or leaves of the plant Glycyrrhiza glabra.
 31. Amethod as claimed in claim 28 wherein the anti-infective agent is ananti-bacterial agent anti-fungal agent or anti-cancer agent.
 32. Amethod as claimed in claim 31 wherein the anti-bacterial agent isselected from the group consisting of β-lactams, macrolides, quinolones,fluoro-quinolones, aminoglycosides, glycopeptides, rifamycins, folateinhibitors, tetracyclines, anti-tuberculosis agents and microbicidalcompounds.
 33. A method as claimed in claim 31 wherein concentration ofglycyrrhizin ranges from 0.05 to 50% the weight of the anti-bacterialagent.
 34. A method as claimed in claim 32 wherein the quinolones andfluoroquinclones are selected from the group consisting of nalidixicacid, norfloxacin, ciprofloxacin, and sparfloxacin.
 35. A method asclaimed in claim 32 wherein the β-Lactams are selected from the groupcomprising penicillin, flucloxacillin, cloxacukkub, methicillin,cephalosporins and carbapenenms.
 36. A method as claimed in claim 32wherein the macrolides are selected from the group consisting oferythromycin, clarithromycin, and azithromycin.
 37. A method as claimedin claim 32 wherein the aminoglycosides are selected from the groupconsisting of streptomycin, gentamicin, and amikacin.
 38. A method asclaimed in claim 32 wherein the glycopeptides are selected fromvancomycin, and teicoplanin.
 39. A method as claimed in claim 32 whereinthe rifamycins are rifampicin.
 40. A method as claimed in claim 32wherein the anti-tuberculosis agents selected from the group consistingof comprising isoniazid, pyrazinamide, and ethambutol.
 41. A method asclaimed in claim 31 wherein concentration of glycyrrhizin ranges from0.25 to 20% the weight of the anti-fimgal agent.
 42. A method as claimedin claim 31 wherein the anti-fungal agent is selected from the groupconsisting of polyene, triazole, imidazole, and clotrimazole.
 43. Amethod as claimed in claim 28 wherein the anti-cancer agent is selectedfrom the group consisting of paclitaxel, docetaxel, vinblastinevincristine, and vinorelbine.
 44. A method as claimed in claim 31wherein the concentration of glycyrrhizin ranges from 10 to 10,000 foldof the weight of the anti-cancer agent.
 45. A method as claimed in claim28 wherein the nutraceutical compound is selected from the groupconsisting of vitamins, amino acids, hormones and other nutritionalsupplements.
 46. A method as claimed in claim 28 wherein administrationof the composition circumvents the side effects of chemotherapy bysubstantially reducing the dosage of the anti-infective or anti-canceragent.
 47. A method as claimed in claim 28 wherein the bioenhancerimproves the uptake of anti-infectives, anticancer agents and othermolecules when glycyrrhizin is provided prior to the treatment withthese molecules.
 48. (canceled)
 49. A method as claimed in claim 46,wherein the side effect is bone marrow suppression, liver toxicity,kidney toxicity, pulmonary scarring, high fever, skin reaction, nausea,vomiting, hair loss, skin rash, mouth sores, diarrhea, loss of appetite,confusion, lethargy, ambulation difficulty, ataxia, an anaphylacticreaction, fast heart rate, wheezing, lowered blood pressure, facialedema, cerebellar dysfunction, slurring of speech, walking problems, eyemotion problem, loss of periods, menses, ovarian failure, soft tissueulcers, cardiotoxicity, a hypersensitivity reaction, abnormal bloodpressure, sweating, bleeding, or shortness of breath.